Dissemination of carbapenemase-producing Enterobacterales in the community of Rawalpindi, Pakistan

Carbapenems are considered last-line beta-lactams for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, their activity is compromised by the rising prevalence of carbapenemase-producing Enterobacterales (CPE), which are especially marked in the Indian subcontinent. In Pakistan, previous reports have warned about the possible spread of CPE in the community, but data are still partial. This study was carried out to analyse the prevalence of CPE, the genetic characterisation, and phylogenetic links among the spreading CPE in the community. In this cohort study, we collected 306 rectal swabs from patients visiting Benazir Bhutto hospital, Rawalpindi. CPEs were screened by using ertapenem-supplemented MacConkey agar. Identification was performed by using conventional biochemical tests, and genomes were sequenced using Illumina chemistry. Antibiotic resistance genes, plasmid incompatibility groups, and Escherichia coli phylogroups were determined in silico. Sequence types were determined by using MLST tool. The prevalence of CPE carriage observed was 14.4% (44/306 samples). The most common carbapenemase-encoding gene was bla-NDM-5 (n = 58) followed by blaNDM-1 (n = 7), blaNDM (non-assigned variant, n = 4), blaOXA-181 (n = 3), blaOXA-232 (n = 3) and blaNDM-7 (n = 1). Most of the CPE were E. coli (55/64, 86%), and the genomic analysis revealed a pauciclonal diffusion of E. coli with ST167 (n = 14), 405 (n = 10), 940 (n = 8), 648 (n = 6) and 617 (n = 5). We obtained a second sample from 94 patients during their hospital stay in whom carriage was negative at admission and found that 7 (7.4%) acquired a CPE. Our results indicate that the prevalence of CPE carriage in the Pakistani urban community was high and driven by the dissemination of some E. coli clones, with ST167 being the most frequent. The high CPE carriage in the community poses a serious public health threat and calls for implementation of adequate preventive measures.

Resistance to antibiotics in Enterobacterales has been on the rise since the past two decades due to the large diffusion of CTX-M-type extended spectrum beta-lactamases (ESBL) which confer resistance to most β-lactams except for carbapenems [9]. Accordingly, the latter are commonly used as last line antibiotics for treating infections caused by multidrug resistant Enterobacterales including those producing ESBLs. Hence, the use of carbapenems has grown in parallel to the rise of ESBL-producing Enterobacterales, paving the emergence and dissemination of carbapenem resistance. While Enterobacterales can resist to carbapenems by combining the production of ESBL and decreased outer-membrane permeability, the main mechanism for carbapenem resistance is the production of carbapenemases [10,11]. The major carbapenemases in Enterobacterales belong to three classes, Ambler class A (e.g., Klebsiella pneumoniae carbapenemase, KPC), class B (e.g. New Delhi metallo β-lactamase, NDM) and class D (e.g. OXA-48 and its close relatives such as OXA-181) [12]. Among them, NDM is one of the most commonly identified carbapenemase worldwide and many studies have reported its high prevalence in the Indian subcontinent [13][14][15]. Moreover, carbapenemaseencoding genes are carried on plasmids which often harbour multiple other antibiotic resistance genes (ARGs), narrowing the range of treatments possibly active on CPE [16].
E. coli and Klebsiella pneumoniae are the most frequent carriers of bla NDM , with specific sequence types (ST) such as ST11, 14, 15, 147 for K. pneumoniae and ST167, 410, 617 for E. coli. Diverse variants of carbapenemases such as VIM, IMP, NDM, KPC, and GIM have also been reported in Enterobacterales across Pakistan [17][18][19]. Limited data are available regarding the epidemiology of CPE carriage in the community [20][21][22], but suggesting a marked dissemination of CPE in Pakistan, Our study aimed to fill these gaps in assessing the CPE carriage prevalence in a Pakistani urban community and the underlying genetic determinants responsible for emergence of carbapenem resistance.

Ethics
The study was approved by the Ethical Review Committee, Rawalpindi Medical University on September 23, 2017. Verbal informed consent was obtained from all participants before the collection of samples, and it was written in participant's record. Patients who refused to participate in the study were excluded. Participation was voluntary, and no minor was included in our study.
Rectal swabs (Nuova, Canelli, Italy) were collected within 24 hours of admission. Patients in whom the first swab was negative for carbapenem-resistant Enterobacterales (CRE), a second swab was taken 72 hours after admission.

Bacterial isolation and identification
We initially did experiments to set the optimum culture conditions to recover CPE with respect to the epidemiological specificities of Pakistani CPE. We cultured already characterised, 4 strains of E. coli (2 each ertapenem-sensitive and ertapenem-resistant) [23] on MacConkey agar (Oxoid, Basingstoke, England) with increasing ertapenem (Merck & Co., Whitehouse Station, USA) concentrations ranging from 0.125 mg/L to 5 mg/L. We finally set the ertapenem concentration at 0.5 mg/L as the optimum concentration at which only resistant bacteria were growing while sensitive were inhibited.

Molecular methods
Genomic DNA of 78 CRE isolates based on resistance pattern of antimicrobial susceptibility profile was extracted using the EZ1 DNA Tissue Kit (Qiagen, Courtaboeuf, France). Libraries were prepared by using Nextera DNA Flex library preparation kit (Illumina, SanDiego, CA). The DNA was quantified using the Qubit Fluorometer (Thermo Fischer Scientific, Asnières sur Seine, France). Whole genome sequencing was performed on NextSeq 550 system (Illumina) with a mid-output kit (2x150 bases). Plasmids DNA of three different E. coli ST167 isolates were extracted with the Plasmid Midi Kit (Qiagen) for MinION system sequencing (Oxford Nanopore Technologies [ONT], Oxford, UK). Libraries were prepared with the Rapid Barcoding Kit, SQK-RBK004 (ONT) then plasmids DNA sequencing was performed on a Flongle Flow Cell (R9.4.1) on a MinION device (ONT).

Multilocus sequence typing (MLST)
MLST profiles were checked by using MLST tool (v2. 16 . A plasmid was considered to be present in a genome if the Illumina reads covered >94% of the plasmid sequence with 10X depth and with maximal mapping quality. The 94% threshold was set according to the lowest coverage observed for the mapping of Illumina reads against the plasmid sequence within the same strain.

Quantification of CRE
Rectal samples thoroughly mixed with 1 mL normal saline were serially diluted till 10 −6 and plated onto MacConkey agar with 0.5 μg/mL ertapenem and without ertapenem and incubated overnight. Colony-forming units (CFU) were counted in decimal logarithms at the dilution in which 1 to 100 CFU were grown. The CRE relative abundance (CRE-RA) was calculated as the ratio of the CRE counts divided by the total number of Gram-negative bacteria, expressed as a percentage. For patients who were found to carry more than one CRE clone, the CRE-RA of the dominant clone was considered.

Statistical analysis
Statistical analysis of relative abundance was performed using Wilcoxon and Kruskal-Wallis tests [42]. Differences of CRE-RA between community-acquired CRE and hospital-associated CRE were calculated by using Wilcoxon test, while Kruskal-Wallis test was used to calculate differences between community-acquired bacterial species and between the different community-acquired E. coli sequence types [43].

Sampling
A total of 306 rectal swabs (one per patient) were collected within 24 hours of their admission to the traumatology and gynaecology wards of Benazir Bhutto hospital. Among them, 27.8% (n = 85) samples were from male patients from the traumatology ward, 21.9% (n = 67) from female patients from the traumatology ward and 50.3% (n = 154) were from female patients from the gynaecology ward. The average age for male and female trauma patients was 38 years and 44 years respectively, while for female gynaecology patients the average age was 28 years. From 94 patients who were subsequently hospitalised and in whom the first swab was negative on the ertapenem-supplemented MacConkey agar, a second swab was taken after 72 hours of their hospital stay (Fig 1). The mean duration between admission and follow-up sampling was eight days (range 3-60 days). Most of the swabs were collected during three periods of time: January to May 2018 (70/306, 23%), August to November 2018 (104/306, 34%), January to May 2019 (132/306, 43%) (Fig 2).

Phenotypic characteristics of the recovered isolates
Enterobacterales isolates were Gram-negative, pink colored, short rods under the microscope. On MacConkey agar E. coli colonies appeared as pink, flat, dry and non-mucoid surrounded with darker pink zone of precipitated bile salts, K. pneumoniae were mucoid, and pink. C. freundii, and E. cloacae also formed pink colonies due to lactose fermentation. Biochemical tests results were interpreted according to Clinical Microbiology procedures handbook [24].

Prevalence of CPE carriage
A total of 170 Enterobacterales isolates was cultured on ertapenem-supplemented MacConkey agar, among which 78 were left after deduplication (based on their antibiotic susceptibility profile). After genomic analysis, 73 were found to produce at least one carbapenemase-encoding gene. Among the 306 patients screened at admission, 48 were community acquired CRE, and 44 carried a CPE, yielding a CPE carriage rate of 14.4% (44/306) in the community (Fig 1). The carriage rate was 16.5% (14/85) among traumatology-admitted male patients, 19.4% (13/ 67) among traumatology-admitted female patients and 11.0% (17/154) among gynaecologyadmitted female patients. All samples were from patients who were not showing any infectious disease symptoms and they were not receiving any medication at the time of sampling.

Antimicrobial susceptibility testing
All the community-carried (n = 69) and hospital-acquired CPE isolates (n = 9) were found to be resistant to ceftazidime, cefotaxime, ertapenem, and cefoxitin while 1 strain (231A) remained apparently intermediate to cefepime. Imipenem was inactive against 92% strains while 79% isolates were resistant against aztreonam. For quinolones, 99% resistance was found against nalidixic acid and 97% against ofloxacin. Trimethoprim/sulfamethoxazole was found resistant against 94% strains, while a lower rate of 24% and 31% resistance was observed for amikacin and gentamicin, respectively. Only 1 isolate (201A) found resistant to colistin (S1 Table), MIC measured for this isolate was 4 mg/L and it was found phenotypically resistant to all antibiotics used except imipenem and amikacin. Bacterial isolates were grouped into extensively-drug resistant (XDR) and multi-drug resistant (MDR) based on the resistance phenotypes definitions of Magiorakos et al. [44]. According to these definitions, none of the isolates was PDR, while 64 (82%) isolates were grouped into XDR and 14 (18%) isolates into MDR category (S2 Table).

Phylogeny
The core genome of the 68 E. coli was made of 2,094 genes (1,807,969 bases in total). The five most frequent STs of community-acquired E. coli made distinct clades (Fig 8). The maximal genetic distances between isolates of the same clade were 1,954 SNPs for ST167, 1,606 SNPs for ST405, 3,098 SNPS for ST940, 3,101 SNPs for ST617 and 3,679 SNPs for ST648. Among all the E. coli, the most genetically distant were separated by 37,852 SNPs. Phylogenetic analysis revealed that community and hospital acquired were closely related isolates suggesting that the community reservoir of CPE is fueling the hospital reservoir. Among ST940 and ST167 community and hospital acquired isolates were clonal sharing less than 10 SNPs.

Replicon typing and plasmid analysis
We identified 27 plasmid replicons in the 78 genomes. The IncX3 and IncFII incompatibility groups commonly associated with bla NDM were present in 23% (18/78) and 41% (32/78) strains, respectively (Fig 9). To assess the diversity of plasmids within the ST167 strains, we sequenced and reconstructed plasmids from three different ST167 E. coli isolates (46D, 60E, and 279D) that had different genetic backgrounds based on the resistance genes, phylogenetics and plasmids typing results. They were found to carry bla NDM on three different IncF plasmids and isolate 46D possessed bla NDM -encoding IncF plasmid with another bla NDM -encoding IncN replicon plasmid. Then, we mapped the Illumina reads of all the E. coli ST167 on the reconstructed plasmids sequences. The four reconstructed plasmids were also found in the genomes of the other E. coli ST167 (Fig 10).

Discussion
The main result of this study is the high prevalence of CPE carriage (14.4%, 44/306) among patients attending the traumatology and gynaecology wards of the Benazir Bhutto hospital of Rawalpindi. This high prevalence rate is in line with previous reports [20,21,45]. High community carriage can lead towards longer hospitalization, higher medical cost, and increased mortality due to limited treatment options. Our study results suggest that community carriage is also one of the important factors for CPE dissemination in hospital settings. A second sample was taken from 94 patients (negative at admission) during their hospital stay and it was found that 7 (7.4%) acquired a CPE. Phylogenetic analysis revealed that there is genetic relatedness of hospital acquired isolates with few community acquired isolates suggesting a person to person spread or same source of contamination. Same clones were circulating in community and hospital settings. In hospital environment, antimicrobial exposure, longer hospitalization, the use of invasive devices, and severe underlying infections are considered independent risk factors for carbapenem producing Enterobacterales, but our study shows increasing CPE carriage among community along with the detailed genomic analysis to understand the determinants responsible for the spread of CPE specially in community settings. Different studies

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including a review of 10 studies of different regions (Asia, Europe, and North America) reported prevalence of CRE ranging from 0.4% to 29.5% isolated from various samples including faecal samples from healthy individuals and outpatients showing that community-based CPE are increasing in Asia, particularly in Indian subcontinent [46][47][48]. In 2013, Nair et al, from India reported 12.3% prevalence of carbapenem resistance among Enterobacterale isolates from patients admitted to different wards, ICU, and visiting outpatient department in respective hospital [49]. Two main factors underlying the specific situation of CPE in Pakistan may be proposed. The first one is the uncontrolled use of antibiotics, several antibiotics can be purchased over-the-counter without prescription, thereby promoting self-medication and potential overuse, causing a big challenge to community health [50]. The second factor is that Pakistan is a developing country where access to clean drinking water and safe food is not secured for large part of population, which may facilitate the circulation of intestinal bacteria such as CPE. A study conducted in Pakistan on environmental water collected from different sites reported the presence of NDM-1 producing bacteria in water sources [51]. In India, the bla NDM was similarly found in tap water [52].

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The diffusion of NDM in the community of Rawalpindi was driven by a limited number of E. coli STs, with 43/55 (78%) of NDM-producing E. coli belonging to the ST167, ST405, ST940, ST648, and ST617. The most frequent ST was ST167 which belongs to the phylogroup A. ST167 NDM-producing E. coli had already been spotted [53][54][55], yet the extent of its diffusion in this region had not been assessed to date. An Indian study conducted in 2020, on sequence

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types of cefotaxime resistant E. coli showing that ST167 was more prevalent in the community followed by ST410 and ST648 [56]. Another study conducted in sewage from Islamabad showed that ST648 was among prevalent STs in E. coli isolates [22]. A Chinese study analysed all bacterial genomes in the GenBank database containing bla NDM [13]. Among these

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genomes, E. coli was the most common species (n = 305) and the ST167 was the main ST (44/ 305) among those possessing bla NDM . In addition, E. coli strains of ST167 carrying bla NDM have been reported from China (hospital-outbreak), India, South Korea, Switzerland, South Africa, and the United States [13,57]. ST167 carrying bla NDM-5 has been the most prevalent sequence type reported from China in clinical settings and has potential to become the main ST type worldwide [58,59].
Combination of resistance genes, and ability to easily disseminate put E. coli ST167 as a potential "high-risk clone" [60] such as the CTX-M-15-producing E. coli ST131. Moreover, using nanopore sequencing technology, we were able to retrieve circularised plasmids for E. coli ST167 strains and among these isolates, we observed several bla NDM -encoding plasmids. The diversity of resistance plasmids observed within ST167 isolates indicates that carbapenem resistance is not spreading through a specific plasmid and that such as E. coli ST131, there does not seem to be a bottleneck for the bla NDM -bearing plasmids for the E. coli ST167.
The genomic analysis showed that bla NDM (90%) was the most prevalent carbapenemase gene in our study and bla NDM-5 was the most common variant (58/70, 82%). Previously, a study from Lahore (Pakistan) showed the presence of bla NDM-5 in isolates from neonates [61]. However, majority of studies showed that bla NDM-1 was more prevalent in Asia especially Indian subcontinent [13,51,57]. Bla NDM-5 seems to be becoming a prevalent NDM variant in the Indian subcontinent [14]. Apart from NDMs, the OXA-181 carbapenemase was found in three community acquired C. freundii belonging to ST107 from three different patients. These three strains were co-harbouring bla NDM-1 and bla OXA-181 and their resistance genes and plasmid content were also similar. Previous studies in Pakistan have reported the presence of the mcr-1 gene in migratory birds, clinical settings, and healthy broilers [62][63][64]. We report here the first detection of mcr-1 from an outpatient in Pakistan. In countries like Pakistan, where the prevalence of CPE is high, increasing resistance against last-line antibiotics such as colistin is of major concern. Spread of mcr genes in CRE is a potential concern as it could create CRE strains that would be potentially pan-drug resistant. Although only one CRE isolate in this

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study was found to be carrying colistin resistance gene, it signifies that surveillance and monitoring should be conducted on regular basis to slow down the emergence of such strains. A study from Thailand also reported similar findings of mcr-1 detection from clinically isolated CRE during 2016-2019 [65]. Dissemination of mcr-1 together with bla NDM in Asian counties is alarming and it needs attention.
The average age for male and female trauma patients was 38 years and 44 years respectively, while for female gynaecology patients the average age was 28 years. According to a study age >50 years is one of the risk factors for carbapenem resistance [66]. Another study has reported high association of carbapenem resistance acquisition in children with age less than ten years [67]. Hence, we assessed the prevalence of CPE in a group with no specific risk factor for multidrug resistance carriage. There is possibility that the CPE prevalence could even be higher in specific populations such as young children or elderly people.
The mean relative abundance of CRE (CRE-RA) was similar to that found in other studies in Europe for community ESBL-producing E. coli [68,69] highlighting that in patients with no recent exposure to antibiotics, multidrug-resistant Enterobacterales remain subdominant. Surprisingly though, the mean CRE-RA of hospital-acquired CRE was not higher. Still, we did not record the antibiotic intake after admission in these patients.
This study will be helpful in raising awareness among medical professionals, scientific community, and policy makers about recent antimicrobial resistance trend in community and need for solutions by limiting the use of antimicrobials and switching towards alternative options such as, vaccines, immunotherapeutics, and modulation of gut microbiota [70].
This work has certain limitations. The population considered might not be a true reflection of community, even though we only included patients who reported no antibiotic intake nor hospitalisation in the 3-months preceding the visit. Also, we used rectal swabs instead of faeces for feasibility matters which may have resulted in underestimation of CPE carriage. Accordingly, we may have overestimated the hospital acquisition rate in that some patients deemed negative for CPE at admission may have been false negative. Screening on ertapenem supplemented MacConkey agar may have led to underestimation of total Enterobacterales as a substantial number of phenotypically susceptible (may carry genotypic resistance markers) strains may have been excluded.

Conclusion
We observed a high carriage rate of CPE among community patients in Rawalpindi, mostly driven by a limited number of E. coli STs among where ST167 was dominant. Asymptomatic CPE carriers are the potential source of its dissemination in community, hospitals as well as in environment as phylogenetic analysis indicated that same CPE clones were circulating in community and hospital settings. Successful dissemination of Enterobacterales clones in community, continuous transmission of carbapenemase harboring plasmids through mechanism of horizontal gene transfer, and co-occurrence of multi-drug resistance genes, are the main reasons for increased carbapenem resistance in community. Stopping dissemination is important otherwise, CPE will achieve a foothold in community and hospitals. CPE transmission can be controlled by active surveillance, raising awareness, regular screening, and implementation of infection control strategies.
Supporting information S1